Denosumab ELISA

KBI1026, Ver1.0 www.eaglebio.com 1

REF

Denosumab ELISA

: KBI1026

Ver 1.0

RUO

Enzyme Immunoassay for the quantitative determination of Denosumab in serum, plasma and cell culture supernatant

RUO REF Catalog Number

LOT Batch Code

Biological Risk

For Research Use Only

Store At

Manufactured By

Expiry Date Consult Operating Instructions

For Research Use Only. Purchase does not include or carry the right to resell or transfer this product either asa stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Eagle Biosciences, Inc. is strictly prohibited.

KBI1026 96 tests

Eagle Biosciences, Inc. 20A NW BLVD., Suite 112Nashua, NH 03063 Phone: +1 (617) 419-2019 Fax: +1 (617) 419-1110 Info@eaglebio.com

REF

Denosumab ELISA

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Introduction:

Denosumab (trade names Prolia and Xgeva) is a human monoclonal antibody for the treatment of osteoporosis, treatment-induced bone loss, metastases to bone, and giant cell tumor of bone. Denosumab is contraindicated in people with low blood calcium levels. The most common side effects are joint and muscle pain in the arms or legs. Denosumab is a RANKL inhibitor, which works by preventing the development of osteoclasts which are cells that break down bone (bone resorption).

Intended Use:

The Eagle Biosciences Denosumab ELISA is used as an analytical tool for quantitative determination ofDenosumab in serum, plasma and cell culture supernatant.

Principle:

The method employs the quantitative sandwich enzyme immunoassay technique. Antibodies to Denosumab are pre-coated onto microwells. Samples and standards are pipetted into microwells and human Denosumab present in the sample are bound by the capture antibody. Then, a HRP (horseradish peroxidase) conjugated anti-Denosumab antibody is pipetted and incubated. After washing microwells in order to remove any non-specific binding, the ready to use substrate solution (TMB) is added to microwells and color develops proportionally to the amount of Denosumab in the sample. Color development is then stopped by addition of stop solution. Absorbance is measured at 450 nm.

Materials Provided:

1. Anti-Denosumab Coated Microtiter Plate (12x8 wells) – 1 no2. Denosumab Standard, (0.5 ml/vial) – 0, 2.5, 5, 10, 20, 40, 80 and 160 ng/ml3. Anti-Denosumab:HRP Conjugate – 12 ml4. Assay Diluent – 6 ml5. Sample Diluent – 50 ml6. Wash Buffer (20X) – 25 ml7. TMB Substrate – 12 ml8. Stop Solution – 12 ml9. Instruction Manual

Materials to be provided by the End-User:

1. Microtiter Plate Reader able to measure absorbance at 450 nm.2. Adjustable pipettes and multichannel pipettor to measure volumes ranging from 25µl to 1000µl3. Deionized (DI) water4. Wash bottle or automated microplate washer5. Semi-Log graph paper or software for data analysis6. Timer7. Absorbent Paper

Handling/Storage:

1. All reagents should be stored at 2ºC to 8ºC for stability.2. All the reagents and wash solutions should be used within 12 months from manufacturing date.3. Before using, bring all components to room temperature (18-25ºC). Upon assay completion ensure all

components of the kit are returned to appropriate storage conditions.

Denosumab ELISA

KBI1026, Ver1.0 www.eaglebio.com 3

4. The Substrate is light-sensitive and should be protected from direct sunlight or UV sources.

Health Hazard Warnings:

1. Reagents that contain preservatives may be harmful if ingested, inhaled or absorbed through the skin.2. For Research Use Only.

Sample Preparation and Storage:

Blood is taken by venipuncture. Serum is separated after clotting by centrifugation. Plasma can be used, too. Lipaemic, hemolytic or contaminated samples should not be run. Repeated freezing and thawing should be avoided. If samples are to be used for several assays, initially aliquot samples and keep at - 20°C.

For Cell Culture Supernatant – If necessary, centrifuge to remove debris prior to analysis. Samples can be

stored at -20°C or -80⁰C. Avoid repeated freeze-thaw cycles.

Preparation Before Use: Allow samples to reach room temperature prior to assay. Take care to agitate patient samples gently in order to ensure homogeneity.

Test Sample preparation - Samples have to be diluted 1:500 to 1:1000 (v/v), e.g. for 1:500 (1 µl sample + 499 µl sample diluent) prior to assay. The samples may be kept at 2 - 8°C for up to three days. Long-term storage requires -20°C.

Reagent Preparation (all reagents should be diluted immediately prior to use):

1. Label any aliquots made with the kit Lot No and Expiration date and store it at appropriate conditionsmentioned.

2. Bring all reagents to Room temperature before use.3. To make Wash Buffer (1X); dilute 50 ml of 20X Wash Buffer in 950 ml of DI water.

Procedural Notes:

1. In order to achieve good assay reproducibility and sensitivity, proper washing of the plates to removeexcess un-reacted reagents is essential.

2. High Dose Hook Effect may be observed in samples with very high concentrations of Denosumab. HighDose Hook Effect is due to excess of antibody for very high concentrations of Denosumab present in thesample. High Dose Hook effect is most likely encountered from samples early in the purification process. IfHook Effect is possible, the samples to be assayed should be diluted with a compatible diluent. Thus if theDenosumab concentration of the undiluted sample is less than the diluted sample, this may be indicativeof the Hook Effect.

3. Avoid assay of Samples containing sodium azide (NaN3), as it could destroy the HRP activity resulting inunder-estimation of the amount of Denosumab.

4. It is recommended that all Standards and Samples be assayed in duplicates.5. Maintain a repetitive timing sequence from well to well for all the steps to ensure that the incubation

timings are same for each well.6. If the Substrate has a distinct blue color prior to use it may have been contaminated and use of such

substrate can lead to compromisation of the sensitivity of the assay.7. The plates should be read within 30 minutes after adding the Stop Solution.8. Make a work list in order to identify the location of Standards and Samples.

Assay Procedure:

1. It is strongly recommended that all Controls and Samples be run in duplicates or triplicates. A standardcurve is required for each assay. All steps must be performed at 37°C

2. Pipette out 50 µl of Assay Diluent in each well.

3. Add 100 µl of Standards or Samples into the respective wells.

Denosumab ELISA

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4. Cover the plate and incubate for 60 minutes at 37°C

5. Aspirate and wash plate 4 times with Wash Buffer (1X) and blot residual buffer by firmly tapping plateupside down on absorbent paper. Wipe of any liquid from the bottom outside of the microtiter wells as anyresidue can interfere in the reading step.

6. Pipette without delay in the same order 100 µl of Anti-Denosumab: HRP Conjugate into each well.

7. Cover the plate and incubate for 60 minutes at 37°C

8. Aspirate and wash plate 4 times with Wash Buffer (1X) and blot residual buffer by firmly tapping plateupside down on absorbent paper. Wipe of any liquid from the bottom outside of the microtiter wells as anyresidue can interfere in the reading step.

9. Add 100 µl of TMB Substrate in each well.

10. Incubate the plate at 37°C for 30 minutes in dark. DO NOT SHAKE or else it may result in higherbackgrounds and worse precision. Positive wells should turn bluish in color.

11. Pipette out 100 µl of Stop Solution. Wells should turn from blue to yellow in color.

12. Read the absorbance at 450 nm with a microplate reader.

Calculation of Results:

Determine the Mean Absorbance for each set of duplicate or triplicate Standards and Samples. Using Semi-Log graph paper, plot the average value (absorbance 450nm) of each standard on the Y-axis versus the corresponding concentration of the standards on the X-axis. Draw the best fit curve through the standard points. To determine the unknown Denosumab concentrations, find the unknown’s Mean Absorbance value on the Y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the Denosumab Concentration. If samples were diluted, multiply by the appropriate dilution factor. Software which is able to generate a cubic spline curve-fit is best recommended for automated results.

Note: It is recommended to repeat the assay at a different dilution factor in the following cases: - If the sample absorbance value is below the first standard.- If the absorbance value is equivalent or higher than the 160 ng/ml standard.

Quality Control:

It is recommended that for each laboratory assay appropriate quality control samples in each run to be used to ensure that all reagents and procedures are correct.

Performance Characteristics of the Kit:

This kit has been validated as per EMA/FDA guidelines in line with ICH Code for Harmonization of Biological Assays.

Sensitivity:

Limit Of Detection: It is defined as the lowest detectable concentration corresponding to a signal of Mean of ‘0’ standard plus 2* SD. 10 replicates of ‘0’ standards were evaluated and the LOD was found to be less than 2.5ng/ml

Linearity:

Standards provided in the kit will be used for measuring the linearity range of Denosumab present in matrix.

Precision:

Denosumab ELISA

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Precision is defined as the percent coefficient of variation (%CV) i.e. standard deviation divided by the mean and multiplied by 100. Assay precision was determined by both intra (n=5 assays) and inter assay (n=5 assays) reproducibility on two pools with low (2.5ng/ml), medium (20ng/ml) and high (160ng/ml) concentrations. While actual precision may vary from laboratory to laboratory and technician to technician, it is recommended that all operators achieve precision below these design goals before reporting results.

Pool Intra Assay %CV Inter Assay %CV

Low <10% <10%

Medium <5% <5%

High <5% <5%

Limitations of Method

Healthy individuals should be tested negative by the Denosumab. This kit is for research use only and should not be used as a diagnostic tool.

Safety Precautions: • This kit is for research use only. Follow the working instructions carefully.• The expiration dates stated on the kit are to be observed. The same relates to the stability stated for

reagents• Do not use or mix reagents from different lots.• Do not use reagents from other manufacturers.• Avoid time shift during pipetting of reagents.• All reagents should be kept in the original shipping container.• Some of the reagents contain small amount of sodium azide (< 0.1 % w/w) as preservative. They must not

be swallowed or allowed to come into contact with skin or mucosa.• Source materials maybe derived from human body fluids or organs used in the preparation of this kit were

tested and found negative for HBsAg and HIV as well as for HCV antibodies. However, no known test guarantees the absence of such viral agents. Therefore, handle all components and all patient samples as if potentially hazardous.

• Since the kit contains potentially hazardous materials, the following precautions should be observed- Do not smoke, eat or drink while handling kit material- Always use protective gloves- Never pipette material by mouth- Wipe up spills promptly, washing the affected surface thoroughly with a decontaminant.• In any case GLP should be applied with all general and individual regulations to the use of this kit.

References: Specific method validation and sample analysis approaches for biocomparability studies of denosumab addressing method and manufacture site changes. Pourvasei R, Lee E, Eschenberg M, Patel V, Macaraeg C, Pandya K, Shih J, Ma M, Lee JW, DeSilva B…AAPS J…2013…Springer

Denosumab for the treatment of osteoporosis. Zaheer S, LeBoff M, Lewiecki EM…Expert Opin Drug Metab Toxicol…2015… Taylor & Francis

Comparison of the efficacy, adverse effects, and cost of zoledronic acid and denosumab in the treatment of osteoporosis. Sheedy KC, Camara MI, Camacho PM…Endocr Pract…2015… AACECORP

Pharmacokinetics, pharmacodynamics, safety, and tolerability of single-dose denosumab in healthy Chinese volunteers: A randomized, single-blind, placebo-controlled study… Qian Chen, Chaoying Hu, Yanmei Liu, Rong Song, Wenjing Zhu, Hongxin Zhao, Antonio Nino, Fan Zhang, Yun Liu…PLOS one…2018…PLOS

The pharmacokinetics and pharmacodynamics of denosumab in patients with advanced solid tumours and bone metastases: a systematic review… Winnie Sohn, Mary Ann Simiens, Kelly Jaeger, Shauna Hutton, and Graham Jang… Br J Clin Pharmacol…2014… Wiley-Blackwell

Denosumab mimics the natural decoy receptor osteoprotegerin by interacting with its major binding site on RANKL…Aneta Schieferdecker,Mareike Voigt,Kristoffer Riecken,Friederike Braig,Thorsten Schinke,Sonja Loges,Carsten BokemeyerBoris Fehse,and Mascha Binder…Oncotarget…2014… Impact Journals

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Denosumab ELISA

SCHEMATIC ASSAY PROCEDURE

1. Remove all components, 30 minutes before adding into the assay plate.

30 mins Components Thaw at Room USE NOW (2-8ºC) RT Before Use Temperature (18-24ºC)

2. Avoid repeated cool-thaw of the components as there will be a loss of activity and this can affect the results.

COOL (2-8ºC) THAW (22+4ºC)

Kit Components NO CHANGE IN RESULTS

AVOID THAW (22+4ºC) COOL (2-8ºC) AVOID

3. Pipette 50 µl Assay Diluent into each well.

4. Pipette 100 µl Standards / Samples into the respective wells.

5. Cover plate and incubate for at 37°C.

6. Aspirate and wash wells 4 times with Wash Buffer (1X).

7. Pipette 100 µl Anti Denosumab:HRP into each well.

8. Cover plate and incubate for at 37°C.

9. Aspirate and wash wells 4 times with Wash Buffer (1X).

10. Pipette 100 µl TMB Substrate into each well.

11. Cover plate and incubate for at 37°C.

12. Pipette 100 µl Stop Solution into each well.

13. Read absorbance at 450nm with a microplate reader within of stopping reaction.

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Denosumab ELISA

Typical Example of a Work List

Well # Contents Absorbance at 450nm

Mean Absorbance

ng/ml Denosumab equivalent

1A

2A

zero std

zero std

1B

2B

2.5 ng/ml

2.5 ng/ml

1C

2C

5.0 ng/ml

5.0 ng/ml

1D

2D

10 ng/ml

10 ng/ml

1E

2E

20 ng/ml

20 ng/ml

1F

2F

40 ng/ml

40 ng/ml

1G

2G

80 ng/ml

80 ng/ml

1H

2H

160 ng/ml

160 ng/ml

3A

4A

Sample

3B

4B

Sample

LIMITED WARRANTY

Eagle Biosciences, Inc. warrants its Product(s) to operate or perform substantially in conformance with its specifications, as set forth in the accompanying package insert. This warranty is expressly limited to the refund of the price of any defective Product or the replacement of any defective Product with new Product. This warranty applies only when the Buyer gives written notice to the Eagle Biosciences within the expiration period of the Product(s) by the Buyer. In addition, Eagle Biosciences has no obligation to replace Product(s) as result of a) Buyer negligence, fault, or misuse, b) improper use, c) improper storage and handling, d) intentional damage, or e) event of force majeure, acts of God, or accident.

Eagle Biosciences makes no warranties, either expressed or implied, except as provided herein, including without limitation thereof, warranties as to marketability, merchantability, fitness for a particular purpose or use, or noninfringement of any intellectual property rights. In no event shall the company be liable for any indirect, incidental, or consequential damages of any nature, or losses or expenses resulting from any defective product or the use of any product. Product(s) may not be resold, modified, or altered for resale without prior written approval from Eagle Biosciences, Inc.

For further information about this kit, its application or the procedures in this kit insert, please contact the Technical Service Team at Eagle Biosciences, Inc. at info@eaglebio.com or at 866-411-8023.

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